Abstract
Malaria is one the world’s most widespread lethal diseases. Plasmodium falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi induce human pathology. These species could be differentially diagnosed using the genotyping of cytochrome b, Pfdhfr and RNA 18S. The persistence of P. falciparum, the most lethal parasite, is mainly due to antimalarial drug resistance. Indeed, a few years after the start of the ambitious malaria eradication program in 1960, chloroquine resistance emerged in Asia and spread widely in all the endemic areas. It was associated with genotypes in P. falciparum chloroquine resistance transporter (CVIET, SVMNT, CVMNT, CVIDT, SVIET and CVMET). The use of new drugs such as sulfadoxine-pyrimethamine (SP) leads quickly to SP-resistant parasites associated with genotypes on P. falciparum DiHydroFolate reductase (I51-R59-N108-I164) and P. falciparum DiHydroPteroate synthetase (436-437-580-613). Recently, the delay of parasite clearance has been described with artemisinine (the most efficacious antimalarial drug). This resistance was associated with the K13 propeller genotype. Since malaria species and antimalarial drug resistance markers could be characterized using nucleic acid sequences, genotyping is needed for malarial monitoring of species distribution and antimalarial drug resistance.
Keywords
- Plasmodium parasites
- drug resistance
- diagnostic
- genotyping
1. Introduction
Malaria remains a major public health problem. More than 40% of the world’s population (3.3 billion) live malarial endemic areas in varying degrees. Despite tremendous efforts in the fight, and though this strategy or plan resulted in significantly decreasing the burden in the last 20 years, Malaria still is persistent in nearly 91 countries (Figure 1). In 2016, the overall incidence was 216 million cases among these and 445,000 deaths were recorded [1]. Africa continues to account for 90% of malaria burden. African children under 5 years of age are the most affected. This infectious disease is due to the invasion of

Figure 1.
Countries endemic for malaria in 2000 and 2016. From WHO [
Decisions concerning malaria treatment depend on the identification of the species causing the disease. Traditionally, this diagnosis was based on the microscopic detection of
2. Genotyping for Plasmodium spp. diagnosis
The genotyping of
2.1. PCR using the specificity of 18S RNA
rRNA is one of the ribosome components. Among rRNA, the

Figure 2.
Specificity and sensitivity of the PCR detection assay. (a) Nested PCR amplification for the demonstration of the specificity of the primers employed. Control genomic DNA from
The nested PCR rFAL1 and rFAL2 generate a 205 bp in presence of the
2.2. RT-PCR NASBA 18S rRNA
Nucleic acid sequence-based amplification (NASBA) is a method in molecular biology, which is used to amplify RNA sequences. This novel approach of genotyping, based on the amplification of nucleic acid sequence (real-time QT-NASBA), was developed by Compton [24]. Immediately after its discovery, the NASBA method was used for the rapid diagnosis and quantification of HIV-1 in patients [25]. Some years later, Schooner et al. [26] developed a real-time quantitative nucleic acid sequence-based amplification (real time QT-NASBA) for the detection of
2.3. PCR sequencing cytochrome b
Cytochrome b (

Table 1.
Amplification primers of the cytochrome b gene (

Figure 3.
The tree of relationship of
The first round of PCR (PCR1) and use of the primers DW2/F and DW4/R produce the fragments of 1253 bp. In the second round of PCR (PCR2), primers
2.4. PCR-RFLP from cytochrome b
Due to the specie specificity and diversity of Cytochrome b, it could be digested with the restriction enzyme

Figure 4.
Illustration of a digestion cytochrome fragment using
2.5. PCR-DHFR
Figure 5 shows the equation of transformation of dihydrofolate to tetrahydrofolate. The dihdrofolate reductase is one of the important malaria proteins involved in the plasmodium folate synthesis. This gene is coded in chromosome 4 and is highly conserved between distantly related species, like plasmodium species. Its linker region revealed significantly a higher sequence diversity than the relatively conserved enzymatic diversity. Different species of

Figure 5.
The equation of the DHF-THF reaction. Dihydrofolate reductase is an enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate. This reaction is essential for the de novo synthesis of purines and certain amino acids. This enzyme is essential for rapid growth and is the target for the action of the important antimalarial drugs pyrimethamine and proguanil.
Primer | Species | Sequence (5′–3′) | Annealing (°C) | No. of cycles | Product (bp) |
---|---|---|---|---|---|
Pla-DHFR-F Pla-TS-R | ATGGARSAMSTYTSMGABGTWTTYGA AAATATTGRTAYTCTGGRTG | 50 | 30 | 1000 | |
Pla-DHFR-NF Pla-TS-R | AAATGYTTYATYATWGGDGG AAATATTGRTAYTCTGGRTG | 55 | 35 | 509–587 | |
PF-Lin-F PF-Lin-R | AAAAGGAGAAGAAAAAAATAA AAAATAAACAAAATCATC | 50 | 35 | 160 | |
PM-Lin-F PM-Lin-R | GACCCAAGAATCCCTCCC CCCATGAAGTTATATTCC | 50 | 35 | 177 | |
PV-Lin-F PV-Lin-R | CGGGAGCACTGCGGACAGCG CACGGGCACGCGGCGGGGC | 55 | 35 | 144 | |
PO-Lin-F PO-Lin-R | GACACACAAAATGATGGGGA ATTGTCCTTTCCTTGACTCG | 55 | 35 | 231, 237 or 243 | |
PK-Lin-F PK-Lin-R | CGATGGATATGGATAGTGG CGCGGGAGAGCATTTCCTC | 58 | 35 | 134 |
Table 2.
Primer sequences and the PCR condition for detection of
3. Genotyping for the monitoring of antimalarial drug resistance
3.1. Antimalarial drug resistance
Antimalarial drug resistance is the ability of
3.2. The genotyping of markers associated with antimalarial drug resistance
3.2.1. P. falciparum chloroquine resistance transporter (Pfcrt)

Figure 6.
The predicted structure and representative haplotypes of
Several mutations have been identified in this transporter. The main mutation T76 allows for the abolition of accumulation of the drug chloroquine in the digestive vacuole. The association of mutations in codons 72, 73, 74, 75 and 76 defined different haplotypes. These haplotypes show a spatio-temporal specificity. CVMNK is the wild-type haplotype that is found in chloroquine-sensitive parasites. In Africa, the most prevalent chloroquine-resistance haplotype is the CVIET. It was also found with less prevalence in South America and in Southeast of Asia. Another
For the genotype haplotype 72–76 of
Genes, codons | Primer names | Primers | T°C | Restriction enzyme | Sizes (bp)* |
---|---|---|---|---|---|
CRT72MS | TTTATATTTTAAGTATTATTTATTTAAGTGGA | 55 | 55 + 38 | ||
76-D2 | CAAAACTATAGTTACCAATTTTG | ||||
CRT745MS | TAAGTATTATTTATTTAAGTGTATGTGTCAT | 55 | 53 + 31 | ||
76-D2 | CAAAACTATAGTTACCAATTTTG | ||||
CRT745MS | TAAGTATTATTTATTTAAGTGTATGTGTCAT | 50 | 53 + 31 | ||
76-D2 | CAAAACTATAGTTACCAATTTTG | ||||
Pfcrt-76A | GCGCGCGCATGGCTCACGTTTAGGTGGAG | 55 | 136 + 56 | ||
Pfcrt-76B | GGGCCCGGCGGATGTTACAAAACTATAGTTACC |
Table 3.
Sequences of primer sets and restriction enzymes used to characterize polymorphisms.
Sizes* indicate the sizes of fragments generated after restriction enzyme digestions. T°C= hybridization temperature during PCR program.
3.2.2. Plasmodium falciparum multidrug resistance 1 (Pfmdr1)

Figure 7.
Genetic polymorphisms in
Some isolates exhibit multicopies of
To the genotype Pfmdr1, followed primers and restriction enzymes are used for PCR-RFLP or for PCR followed by sequencing gene. Digestion of PCR products gives fragments of 126 and 165 bp for mutant 86Y whereas wild type N86 is not digested by restriction enzyme
Genes, Codons | Primer names | Primers | T°C | Restriction enzyme | Sizes (bp)* |
---|---|---|---|---|---|
mdr86D1 | TTTACCGTTTAAATGTTTACCTGC | 45 | 126 + 165 | ||
mdr86D2 | CCATCTTGATAAAAAACACTTCTT | ||||
mdr1246D1 | AATGTAAATGAATTTTCAAACC | 45 | 113 + 90 | ||
mdr1246D2 | CATCTTCTCTTCCAAATTTGATA |
Table 4.
Sequences of primer sets and restriction enzymes used to characterize
Sizes* indicate the sizes of fragments generated after restriction enzyme digestions. T°C = hybridization temperature during PCR program.
3.2.3. Plasmodium falciparum dihydrofolate reductase (PfDHFR)

Table 5.
Primer sets and RFLP conditions to genotypes
3.2.4. Plasmodium falciparum dihydropteroate synthase (PfDHPS)
Triple mutant I51R59N108 in
3.2.5. Plasmodium falciparum ATPase 6 (PfATPASE6)
The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, ortholog of
3.2.5.1. P. falciparum multidrug resistance protein (PfMRP)
3.2.5.2. P. falciparum Kelch 13 (PfK13)
Chinese populations used

Figure 8.
Several investigations on K13 genotyping reported that mutations M476I, Y493H, R539T, I543T, P553L and C580Y conferred a greater artemisinin resistance [63]. Other mutations F446I and A578S were described in PfK13. A578S, widespread in Africa, is not associated with artemisinin resistance. These genotypes are investigated by PCR sequencing.
4. Conclusions
Due to the nucleotide specificities of each